Nuclear magnetic resonance (NMR) spectroscopy was used to monitor, on a beat-to-beat basis, the concentration of creatine phosphate and adenosine triphosphate during alterations in the work output of canine hearts in vivo. Over a wide range of rate-pressure products (5,000 to 25,000 mmHg/min), the relative amounts of creatine phosphate and adenosine triphosphate within the heart remained constant. The relative concentration of free adenosine diphosphate was calculated under the reasonable assumption that the creatine kinase-catalyzed reaction is near equilibrium in this tissue. The free concentration of adenosine diphosphate also did not change over this range of rate-pressure products. The data demonstrate that the concentration of these compounds is highly regulated in vivo and suggest that factors other than their concentration may be involved in the modulation of steady-state myocardial work output with oxygen consumption.